Multilevel microfluidic systems and methods

ABSTRACT

Multilevel microfluidic devices include a control line that can simultaneously actuate valves for both sample and reagent lines. Microfluidic devices are configured to contain a first reagent in a first chamber and a second reagent in a second chamber, where either or both of the first and second reagents are contained at a desired or selected pressure. Operation of a microfluidic device includes transmitting second reagent from the second chamber to the first chamber, for mixing or contact with the first reagent. Microfluidic device features such as channels, valves, chambers, can be at least partially contained, embedded, or formed by or within one or more layers or levels of an elastomeric block.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/422,612 (which is now U.S. Pat. No. 8,475,743), filed Apr. 13, 2009,which claims the benefit U.S. Provisional Application No. 61/044,417,filed Apr. 11, 2008. This application is also related to U.S. patentapplication Ser. No. 11/043,895 (which is now U.S. Pat. No. 8,105,553)filed Jan. 25, 2005. The entire content of each of the above-referencedfilings is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Embodiments of the present invention relate to the fields ofmicrofluidics, lab-on-a-chip, Polymerase Chain Reactions (“PCR”),biochemical analysis, protein crystallization and screening for proteincrystallization conditions, microfabrication, laboratory robotics,immunoassays, and automated biological screening and analysis, amongothers.

Microfluidic devices can be defined as devices having one or morefluidic pathways, often called channels, microchannels, trenches, lines,or recesses, having a cross-sectional dimension below 1000 μm, and whichoffer benefits such as increased throughput and reduction of reactionvolumes. Relatedly, there is a continuing trend toward increasing thenumber of reactions that can be performed with a microfluidic device.For example, it is often desirable to provide devices having a highdensity of reaction chambers. Despite significant recent advances inmicrofluidic technology, existing fabrication techniques often presentobstacles which preclude the development of even more efficient devices.

Hence, there remains a continuing need for improved manufacturingmethods for producing microfluidic devices having a higher density ofreaction or detection zones per unit area of the microfluidic device. Atleast some of these objectives will be met by embodiments of the presentinvention.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention provide microfluidic devices havinga high density of reaction chambers or zones per unit area, as well asmethods for their use and manufacture. Such devices can be made smallerthan existing devices, and often provide improved performancecharacteristics. The general benefits of using microfluidic systemsinclude a substantial reduction in time, cost, and space requirementsfor the devices utilized to conduct the analysis or synthesis. Forexample, many diagnostic assays require the use of expensive reagents,and it may be difficult or expensive to obtain large testing samples.Devices which can utilize smaller amounts of reagent and sample are ableto provide more data points at a lower cost. Exemplary embodiments arewell suited for use in crystal formation and amplification reactions. Insome cases, microfluidic devices may have a control line that cansimultaneously actuate valves for both sample and reagent lines.Relatedly, microfluidic devices may be configured to contain a firstreagent in a first chamber and a second reagent in a second chamber,where either or both of the first and second reagents are contained at adesired or selected pressure. In some cases, operation of themicrofluidic device includes transmitting second reagent from the secondchamber to the first chamber, for mixing or contact with the firstreagent. Microfluidic device features such as channels, valves,chambers, can be at least partially contained, embedded, or formed by orwithin one or more layers or levels of an elastomeric block.

In one aspect, embodiments of the present invention encompassmicrofluidic devices having a first flow channel, a second flow channel,and a control channel. The first flow channel can be formed in a firstlayer of an elastomeric substrate, the control channel can be formed ina second layer of an elastomeric substrate, and the second flow channelcan be formed in a third layer of an elastomeric substrate. Often, thesecond layer is adjacent to the first layer, and the third layer isadjacent to the second layer. A change in pressure within the firstcontrol channel can modulate fluid flow within the first and second flowchannels. Microfluidic devices can also include a first isolation valvedisposed along the first flow channel, where the first control channelcontrols operation of the first isolation valve. The first isolationvalve may include a deflectable membrane. Microfluidic devices can alsoinclude a second isolation valve disposed along the second flow channel,where the first control channel controls operation of the secondisolation valve. The second isolation valve may include a deflectablemembrane. Microfluidic devices may also include a first chamber disposedat least partially within the first layer, and a second chamber disposedat least partially within the first layer. The first chamber can be influid communication with the first flow channel. The second chamber canbe in fluid communication with the second flow channel. In someembodiments, a change in pressure within the first control channelsimultaneously modulates fluid flow within the first and second flowchannels.

In another aspect, embodiments of the present invention encompass amicrofluidic device having an elastomeric substrate with multiplelayers. For example, the elastomeric substrate can have a first layer, asecond layer, and a third layer, where the second layer is disposedbetween the first and third layers. The device can also include a firstchamber formed at least partially within the first layer of theelastomeric substrate, and a second chamber formed at least partiallywithin the first layer of the elastomeric substrate. Further, the devicemay include a first control channel formed in the second layer of theelastomeric substrate. Often, the device is configured so that a changein pressure within the first control channel modulates a first fluidflow passing through the first layer and into the first chamber, andalso modulates a second fluid flow passing through the third layer andinto the second chamber. In some instances, the device includes aninterface channel that provides fluid communication between the firstchamber and the second chamber. For example, the interface channel maybe formed in the third layer. In some cases, the interface channel is influid communication with the second flow channel. The device may alsoinclude an interface valve disposed along the interface channel. Theinterface valve may modulate flow through the interface channel betweenthe first and second chambers. In some embodiments, the interface valvecomprises a deflectable membrane.

In one aspect, embodiments of the present invention provide amicrofluidic device. The device can include a first flow channel formedin a first layer of an elastomeric substrate, a first chamber in fluidcommunication with the first flow channel, and a first isolation valvedisposed along the first flow channel. The first isolation valve caninclude a first portion of a control channel formed in a second layer ofthe elastomeric substrate adjacent to the first layer. The firstisolation valve can be configured to control flow through the first flowchannel into the first chamber. The device may also include a secondflow channel formed in a third layer of the elastomeric substrateadjacent to the second layer, a second chamber in fluid communicationwith the second flow channel, and a second isolation valve disposedalong the second flow channel. The second isolation valve can include asecond portion of the control channel formed in a second layer of theelastomeric substrate. The second isolation valve can be configured tocontrol flow through the second flow channel into the second chamber.The device can also include a reaction channel formed in the third layerof the elastomeric substrate, in fluid communication with the firstchamber and the second chamber, and an interface valve disposed alongthe reaction channel between the first and second chamber. The interfacevalve can include a portion of an interface channel formed in a fourthlayer of the elastomeric substrate adjacent to the third layer, and canbe configured to control flow through the reaction channel. In someembodiments, the first isolation valve includes a deflectable membrane.In some embodiments, the second isolation valve includes a deflectablemembrane. In some embodiments, the interface valve includes adeflectable membrane. The first chamber can define a first chambervolume, the second chamber can define a second chamber volume. In somecases, the first chamber volume is less than the second chamber volume.In some cases, the first chamber volume is greater than the secondchamber volume. In some cases, the first chamber volume is equal to thesecond chamber volume.

In another aspect, embodiments of the present invention encompassmethods of mixing or reacting materials in a microfluidic device. Anexemplary mixing technique includes flowing a first material through afirst flow channel formed in a first layer of an elastomeric substrate,and flowing the first material through a first isolation valve. Thefirst isolation valve can be disposed along the first flow channel, caninclude a first portion of a control channel formed in a second layer ofthe elastomeric substrate adjacent to the first layer, and can beconfigured to control flow through the first flow channel into a firstchamber. The technique can also include flowing the first material fromthe first flow channel into the first chamber. Further, the mixingprocess can include flowing a second material through a second flowchannel formed in a third layer of the elastomeric substrate adjacent tothe second layer, and flowing the second material through a secondisolation valve. The second isolation valve can be disposed along thesecond flow channel, can include a second portion of the control channelformed in a second layer of the elastomeric substrate, and can beconfigured to control flow through the second flow channel into thesecond chamber. The mixing procedure can also include flowing the secondmaterial from the second flow channel into the second chamber, actuatingthe control channel so as to inhibit flow through the first and secondisolation valves, and flowing the first material from the first chamberthrough an interface valve into the second chamber, so as to mix thefirst material with the second material. The interface valve can includea portion of an interface channel formed in a fourth layer of theelastomeric substrate adjacent to the third layer, and can be configuredto control flow between the first chamber and the second chamber. Insome embodiments, a first isolation valve includes a first deflectablemembrane, a second isolation valve includes a second deflectablemembrane, and the process of actuating the control channel includesactuating the first and second deflectable membranes. In someembodiments, mixing techniques can include actuating an interfacechannel to provide fluid communication between the first chamber and thesecond chamber. An interface valve can include a portion of an interfacechannel formed in a fourth layer of the elastomeric substrate adjacentto the third layer, and can be configured to control flow through areaction channel formed in the third layer. In some cases, an interfacevalve can include a deflectable membrane, and the process of actuatingthe interface channel can include actuating the deflectable membrane.Exemplary mixing techniques may also include holding the first materialin the first chamber at first pressure and holding the second materialin the second chamber at a second pressure, prior to flowing the firstmaterial into the second chamber. In some cases, the first pressure isgreater than the second pressure. In some cases, the first pressure canbe about 10 psi and the second pressure can be about 0 psi.

In yet another aspect, embodiments of the present invention include amicrofluidic device having a plurality of first flow channels formed ina first layer of an elastomeric substrate, and a plurality of firstchambers. Each one of the plurality of first chambers can be in fluidcommunication with a corresponding first flow channel of the pluralityof first flow channels. A microfluidic device can also include aplurality of control channels formed in a second layer of theelastomeric substrate adjacent to the first layer, and a plurality offirst isolation valves. Each one of the plurality of first isolationvalves can be disposed along a corresponding first flow channel of theplurality of first flow channels, can include a first portion of acorresponding control channel of the plurality of control channels, andcan be configured to control flow through the corresponding first flowchannel into a corresponding first chamber of the plurality of firstchambers. Further, a microfluidic device can have a plurality of secondflow channels formed in a third layer of the elastomeric substrateadjacent to the second layer, and a plurality of second chambers. Eachone of the plurality of second chambers can be in fluid communicationwith a corresponding second flow channel of the plurality of second flowchannels. A microfluidic device can also have a plurality of secondisolation valves. Each one of the plurality of second isolation valvescan be disposed along a corresponding second flow channel of theplurality of second flow channels, can include a second portion of thecorresponding control channel of the plurality of control channels, andcan be configured to control flow through the corresponding second flowchannel into a corresponding second chamber of the plurality of secondchambers. Still further, a microfluidic device can have a plurality ofreaction channels formed in the third layer of the elastomericsubstrate. Each one of the plurality of reaction channels can be influid communication with a corresponding first chamber of the pluralityof first chambers and a corresponding second chamber of the plurality ofsecond chambers. A microfluidic device may also include a plurality ofinterface valves. Each one of the plurality of interface valves can bedisposed along a corresponding reaction channel of the plurality ofreaction channels between the corresponding first chamber and thecorresponding second chamber, can include a portion of a correspondinginterface channel of a plurality of interface channels formed in afourth layer of the elastomeric substrate adjacent to the third layer,and can be configured to control flow through the corresponding reactionchannel.

In a still further aspect, embodiments of the present inventionencompass a microfluidic device having a first flow channel formed in afirst layer of an elastomeric substrate, a first control channel formedin a second layer of an elastomeric substrate, and a second flow channelformed in a third layer of an elastomeric substrate. The second layercan be adjacent to and between the first layer and the third layer. Themicrofluidic device can be configured so that a change in pressurewithin the first control channel simultaneously modulates fluid flowwithin the first and second flow channels.

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a perspective view of a unit cell of a microfluidicdevice according to embodiments of the present invention.

FIG. 1A shows an exploded perspective view of individual layers of aunit cell of a microfluidic device according to embodiments of thepresent invention.

FIGS. 1B to 1E show microfluidic molds according to embodiments of thepresent invention.

FIG. 2 illustrates a perspective view of unit cell of a microfluidicdevice according to embodiments of the present invention.

FIG. 3 illustrates a perspective view of a microfluidic device matrixhaving multiple unit cells according to embodiments of the presentinvention.

FIG. 4 illustrates a perspective view of a microfluidic device matrixhaving multiple unit cells according to embodiments of the presentinvention.

FIGS. 5A to 5C show cross-section views of a microfluidic device unitcell according to embodiments of the present invention.

FIGS. 6A and 6B show a microfluidic device according to embodiments ofthe present invention.

FIG. 7 illustrates a microfluidic device according to embodiments of thepresent invention.

FIG. 8 illustrates a microfluidic device according to embodiments of thepresent invention.

FIG. 9 illustrates a photomicrograph, with scale bar, of an exemplarymicrofluidic device according to embodiments of the present invention.

FIG. 10 illustrates a reader image of an exemplary microfluidic deviceaccording to embodiments of the present invention.

FIG. 11 illustrates a reader image of an exemplary microfluidic deviceaccording to embodiments of the present invention.

FIG. 12 illustrates a reader image of an exemplary microfluidic deviceaccording to embodiments of the present invention.

FIG. 13 illustrates an exemplary microfluidic device, showing channelconnections between input wells and an elastomeric block, according toembodiments of the present invention.

FIG. 14 illustrates aspects of a microfluidic system and methodaccording to embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the invention is not limited to the particularmethodology, protocols, and reagents, etc., described herein, as thesemay vary as the skilled artisan will recognize. It is also to beunderstood that the terminology used herein is used for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the invention. It is also to be noted that as used herein andin the appended claims, the singular forms “a,” “an,” and “the” includethe plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a cell” is a reference to one or morecells and equivalents thereof known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which the invention pertains. The embodiments of theinvention and the various features and advantageous details thereof areexplained more fully with reference to the non-limiting embodiments andexamples that are described and/or illustrated in the accompanyingdrawings and detailed in the following description. It should be notedthat the features illustrated in the drawings are not necessarily drawnto scale, and features of one embodiment may be employed with otherembodiments as the skilled artisan would recognize, even if notexplicitly stated herein. Descriptions of well-known components andprocessing techniques may be omitted so as to not unnecessarily obscurethe embodiments of the invention. The examples used herein are intendedmerely to facilitate an understanding of ways in which the invention maybe practiced and to further enable those of skill in the art to practicethe embodiments of the invention. Accordingly, the examples andembodiments herein should not be construed as limiting the scope of theinvention, which is defined solely by the appended claims and applicablelaw. Moreover, it is noted that like reference numerals referencesimilar parts throughout the several views of the drawings.

Accordingly, provided immediately below is a “Definition” section, wherecertain terms related to the invention are defined specifically forclarity, but all of the definitions are consistent with how a skilledartisan would understand these terms. Particular methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention. All references referred to herein areincorporated by reference herein in their entirety.

DEFINITIONS

PNA is peptide nucleic acid

LNA is locked nucleic acid

DA is dynamic array

PCR is polymerase chain reaction

BSA is bovine serum albumin

FRET is fluorescence resonance energy transfer

GT is genotyping

PEG is polyethylene glycol

PLP is padlock probe

The term “adjacent” as used herein, generally refers to the positioningof the primer with respect to the probe on its complementary strand ofthe target nucleic acid analyte. The primer and probe may be separatedin a range of about 1 to about 20 nucleotides, more specifically, in arange of about 1 to about 10 nucleotides, or may directly abut oneanother.

The term “analyte” as used herein, generally refers to a nucleic acidmolecule or mixture of nucleic acid molecules, defined infra, that is tobe detected or quantified using the methods of the invention. The terms“target nucleic acid analyte” and “nucleic acid analyte” are usedinterchangeably with the term “analyte” for the purposes of thisinvention.

The terms “complementary” or “complementarity” as used herein, mayinclude the natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of molecules.

The term “covalently attached” as used herein, generally refers to anattachment of one molecular moiety to another molecular moiety throughcovalent chemical bonds.

The term “dye” as used herein, generally refers to any organic orinorganic molecule that absorbs electromagnetic radiation at awavelength greater than or equal 340 nm.

The term “fluorescent dye” as used herein, generally refers to any dyethat emits electromagnetic radiation of longer wavelength by afluorescent mechanism upon irradiation by a source of electromagneticradiation, such as a lamp, a photodiode, or a laser.

The term “GT sample buffer,” as used herein generally refers to a bufferthat is capable of blocking binding sites on the surface of the reactionchannels and chambers in a DA chip. The buffer protects the reactioncomponents from depletion during the chip loading process or reaction.It may also reduce the usage of additional Taq-Gold Polymerase by lessthan about 80% for reagent costs. A 20×GT buffer may include acombination of betaine (FW 117.15), BSA, Superblock® T20 (in PBS)(Thermo Scientific, Rockford, Ill.), Superblock® (in PBS) (ThermoScientific, Rockford, Ill.), Superblock® (in TBS) (Thermo Scientific,Rockford, Ill.), Superblock® T20 (in TBS) (Thermo Scientific, Rockford,Ill.), glycerol, PEG 20,000, PEG MME550, PEG MME5000, and Tween 20.

The term “homogenous assay” as used herein, generally refers to a methodto detect or quantify a nucleic acid analyte that requires no post-assayprocessing to record the result of the assay. The homogenous assays maybe carried out in closed tubes or microfluidic arrays where no furtheraddition of reagents or supplementary chemicals are necessary to recordthe result once the assay is started. Homogenous assays allowrecordation of the result of the assay in real time, meaning that theresult of the assay can be continuously recorded as the assay progressesin time.

The term “hydrolysis probes” as used herein are generally described inU.S. Pat. No. 5,210,015 incorporated herein by reference in itsentirety. Hydrolysis probes take advantage of the 5′-nuclease activitypresent in the thermostable Taq polymerase enzyme used in the PCRreaction (TaqMan® probe technology, Applied Biosystems, Foster CityCalif.). The hydrolysis probe is labeled with a fluorescent detector dyesuch as fluorescin, and an acceptor dye or quencher. In general, thefluorescent dye is covalently attached to the 5′ end of the probe andthe quencher is attached to the 3′ end of the probe, and when the probeis intact, the fluorescence of the detector dye is quenched byfluorescence resonance energy transfer (FRET). The probe may annealdownstream of one of the primers that defines one end of theamplification target site on the nucleic acid target analyte in the PCRreaction. Using the polymerase activity of the Taq enzyme, amplificationof the target nucleic acid analyte is directed by one primer that isupstream of the probe and a second primer that is downstream of theprobe but anneals to the opposite strand of the target nucleic acid. Asthe upstream primer is extended, the Taq polymerase reaches the regionwhere the labeled probe is annealed, recognizes the probe-templatehybrid as a substrate, and hydrolyzes phosphodiester bonds of the probe.The hydrolysis reaction irrevocably releases the quenching effect of thequencher dye on the reporter dye, thus resulting in increasing detectorfluorescence with each successive PCR cycle. In particular, thehydrolysis probes of the invention may capable of detecting 8-mer or9-mer motifs that are common in the human and other transcriptomes andmay have a high T_(m) of about 70° C. enabled by LNA analogs.

The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable and/or quantifiable signal. Inparticular, the label can be attached to a nucleic acid or protein.Labels may provide signals detectable by fluorescence, radioactivity,colorimetric, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like.

The term “nucleic acid” as used herein generally refers to cDNA, DNA,RNA, single-stranded or double-stranded and any chemical modificationthereof, such as PNA and LNA. LNAs are described in U.S. Pat. Nos.6,794,499, 6,670,461, 6,262,490, and 6,770,748 herein incorporated byreference in their entirety. Nucleic acids may be of any size. Nucleicacid modifications may include addition of chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and functionality to the individual nucleicacid bases or to the nucleic acid as a whole. Such modifications mayinclude modified bases such as 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at cytosine exocyclic amines, substitutions of5-bromo-uracil, backbone modifications, methylations, unusual basepairing combinations such as the isobases isocytidine and isoguanidineand the like. The nucleic acid can be derived from a completely chemicalsynthesis process, such as a solid phase mediated chemical synthesis, orfrom a biological origin, such as through isolation from almost anyspecies that can provide nucleic acid, or from processes that involvethe manipulation of nucleic acids by molecular biology tools, such asDNA replication, PCR amplification, reverse transcription, or from acombination of those processes.

The term “nucleic acid probe” as used herein is a nucleic acid thatcarriers at least one covalently attached dye, such as a fluorescentdye. In particular, the probe does not contain a sequence complementaryto sequences used to prime the PCR reaction.

The term “padlock probe” or “PLP” as used herein, generally refers tolinear oligonucleotides having a length of about 100 base pairs. Thesequences at the 3′ and 5′ ends of the PLP are complementary to adjacentsequences in the target nucleic acid analyte. In the central,noncomplementary region of the PLP there is a “tag sequence” that may beused to identify the specific PLP. The tag sequence may be flanked byuniversal primer sites or unique and/or specific primer sites, whichallow PCR amplification of the tag sequence. Upon hybridization to thetarget, the 5′ and 3′ ends of the PLP are brought into close proximityand may be subsequently ligated. The resulting product is a circularprobe molecule catenated to the target nucleic acid analyte. The tagregions of circularized PLPs may be amplified and quantified and/ordetected using TAQMAN® Real Time PCR, for example. The presence andamount of amplicon may be correlated with the presence and quantity oftarget sequence in the sample. For descriptions of PLPs see, e.g.,Landegren et al., 2003, Padlock and proximity probes for in situ andarray-based analyses: tools for the post-genomic era, Comparative andFunctional Genomics 4:525-30; Nilsson et al., 2006, Analyzing genesusing closing and replicating circles Trends Biotechnol. 24:83-8;Nilsson et al., 1994, Padlock probes: circularizing oligonucleotides forlocalized DNA detection, Science 265:2085-8. The above references areincorporated by reference herein in their entirety.

The term “PCR,” as used herein, generally refers to a method foramplifying, detecting, or quantifying a specific region of an analyte.One skilled in the art appreciates that there are several variations onthe basic PCR technique such as allele-specific PCR, assembly PCR orpolymerase cycling assembly (PCA), colony PCR, helicase-dependentamplification, hot start PCR, intersequence-specific (ISSR) PCR, inversePCR, ligation-mediated PCR, methylation-specific PCR, multiplex ligationdependent probe amplification, multiplex PCR, nested PCR,overlap-extension PCR, quantitative PCR, quantitative real-time PCR,RT-PCR, thermal asymmetric interlaces (TAIL) PCR, touchdown PCR, andPAN-AC. Additionally, one skilled in the art would understand how topractice these variations on the basic PCR technique.

The phase “preliminary amplification reaction” as used herein, generallyrefers to processes for preparing the sample prior to running thehomogenous assay. The term “pre-amplified sample” may be usedinterchangeably with the phrase “preliminary amplification reaction” forthe purposes of the invention herein.

The term “purification,” as used herein, generally refers to any processby which proteins, polypeptides, or nucleic acids are separated fromother elements or compounds on the basis of charge, molecular size, orbinding affinity.

The term “quencher” as used herein, generally refers to dye that reducesthe emission of fluorescence of another dye.

The term “querying” as used herein, generally refers to determiningwhether a target-specific probe is associated with (e.g., bound to orcantenated with) the nucleic acid analyte, and optionally quantifyingthe amount of target-specific probe in the sample.

A “sample” as used herein, generally refers to a sample of tissue orfluid from a human or animal including, but not limited to plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal and genitourinary tracts, tears, saliva, bloodcells, tumors, organs, tissue and sample of in vitro cell cultureconstituents. In particular, the sample may be single cells, paraffinembedded tissue samples, and needle biopsies. Moreover, a sample mayinclude environmental samples such as lake water, and food samples.

The phrase “substantially purified,” or “substantially isolated,” asused herein generally includes nucleic or amino acid sequences that areremoved from their natural environment, isolated or separated, and areat least about 60% free, specifically at least about 75% free, and mostspecifically at least about 90% free from other components with whichthey may be associated with, and includes recombinant or cloned nucleicacid isolates and chemically synthesized analogs or analogs biologicallysynthesized by systems.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake elastomeric blocks, layers, membranes, microvalves, pumps, and thelike. Variations in the materials used may in some cases be driven bythe need for particular material properties, i.e. solvent resistance,stiffness, gas permeability, or temperature stability. There are many,many types of elastomeric polymers. A brief description of the mostcommon classes of elastomers is presented here, with the intent ofshowing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones orpolysiloxanes.

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (generally, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Pure polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (.about.1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thepolyisobutylene backbone, which may then be vulcanized as above.

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for a photoresist encapsulation system(where there will be plenty of unreacted monomer in the liquid layerpoured on top of the cured layer). Incomplete curing would allowhomogeneous multilayer soft lithography (A to A bonding). The chemistryalso facilitates making one layer with extra butadiene (“A”) andcoupling agent and the other layer (“B”) with a butadiene deficit (forheterogeneous multilayer soft lithography). SBS is a “thermosetelastomer”, meaning that above a certain temperature it melts andbecomes plastic (as opposed to elastic); reducing the temperature yieldsthe elastomer again. Thus, layers can be bonded together by heating.

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, make them useful forheterogeneous multilayer soft lithography just as RTV 615 is: by usingexcess A-A in one layer and excess B-B in the other layer.

Silicone polymers have great structural variety, and provide a greatnumber of commercially available formulations. The vinyl-to-(Si—H)crosslinking of RTV 615 (which allows both heterogeneous multilayer softlithography and photoresist encapsulation) has already been discussed,but this is only one of several crosslinking methods used in siliconepolymer chemistry.

In addition to the use of the simple “pure” polymers discussed above,crosslinking agents may be added. Some agents (like the monomers bearingpendant double bonds for vulcanization) are suitable for allowinghomogeneous (A to A) multilayer soft lithography or photoresistencapsulation; in such an approach the same agent is incorporated intoboth elastomer layers. Complementary agents (i.e. one monomer bearing apendant double bond, and another bearing a pendant Si—H group) aresuitable for heterogeneous (A to B) multilayer soft lithography. In thisapproach complementary agents are added to adjacent layers.

The following is a non-exclusive list of elastomeric materials which maybe utilized in connection with the present invention: polyisoprene,polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and siliconepolymers; or poly(bis(fluoroaLkoxy)phosphazene) (PNF, Eypel-F),poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(1-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon).

Allcock et al, Contemporary Polymer Chemistry, 2nd Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Materials having a Young's modulus of between about 1 Pa to about 1 TPa,or between about 10 Pa to about 100 GPa, or between about 20 Pa to about1 GPa, or between about 50 Pa to about 10 MPa, or between about 100 Pato about 1 MPa are useful in accordance with embodiments of the presentinvention, although materials having a Young's modulus outside of theseranges could also be utilized depending upon the needs of a particularapplication. In some cases, materials can have a Young's modulus ofabout 100 MPA (megapascals) or less. In other embodiments, the Young'smodulus of the material is about 75 MPA or less, about 50 MPa or less,about 25 MPa or less, about 10 MPa or less, about 8 MPa or less, about 5MPa or less, or about 2 MPa or less.

Embodiments of the present invention provide a microfluidic device thatincludes features such as channels, valves, and chambers, that are atleast partially contained, embedded, or formed by or within one or morelayers or levels of an elastomeric block. An exemplary microfluidicdevice has a reagent flow channel, or reagent line, formed in a firstlayer of an elastomer. The reagent flow channel includes a containmentvalve and a chamber conduit. The microfluidic device may also have acontrol channel, or containment line, formed in a second layer of theelastomer adjacent to the first layer. Further, the microfluidic devicemay contain a sample flow channel, or sample line, formed in a thirdlayer of the elastomer adjacent to the second layer. The sample flowchannel may include a containment valve and a chamber conduit. Thecontrol channel can be in operative association with both the reagentflow channel containment valve and the sample flow channel containmentvalve. The microfluidic device can include a reagent chamber in fluidcommunication with the reagent line, and a sample chamber in fluidcommunication with the sample line. The reagent chamber and the samplechamber may be in fluid communication with each other by way of areaction flow channel or reaction line, formed in the third layer of theelastomer. The reaction line may include an interface valve. Themicrofluidic device may also include an interface channel formed in afourth layer of the elastomer adjacent to the third layer. The interfacechannel can be in operative association with the reaction flow channelinterface valve.

Embodiments of the present invention also encompass methods of makingand using the microfluidic devices disclosed herein. For example,operation of a microfluidic device can involve opening one or moreisolation valves, closing one or more interface valves, and flowingmaterial past the isolation valves and into one or more chambers,optionally under pressure. Techniques may also include changing thepressure in a containment line to close the isolation valves, so as toseal off the individual chambers, and changing the pressure in aninterface line, so as to open an interface valve. A first material in afirst chamber can flow past an open interface valve and into a secondchamber, where the first material mixes or reacts with a second materialcontained therein.

Turning now to the drawings, FIG. 1 depicts a perspective view of a unitcell 100 of a microfluidic device, according to embodiments of thepresent invention. Unit cell 100 includes a first channel 130, a firstisolation valve 132, a first chamber 140, a second channel 110, a secondisolation valve 112, a second chamber 120, a control channel 150, aninterface channel 160, an interface valve 162, and a reaction channel170. Typically, these features are at least partially contained,embedded, or formed by or within an elastomeric block 180. As shownhere, first channel 130 is at least partially disposed within a firstlayer 181 of elastomeric block 180. Control channel 150 is at leastpartially disposed within a second layer 182 of elastomeric block 180,where the second layer is adjacent to the first layer. Second channel110 is at least partially disposed within a third layer 183 ofelastomeric block 180, where the third layer is adjacent to the secondlayer. Interface channel 160 is at least partially disposed within afourth layer 184 of elastomeric block 180, where the fourth layer isadjacent to the third layer.

With reference to the “A” arrows, a first material, such as an assayreagent, can flow through first channel 130, past first isolation valve132, and into first chamber 140. Similarly, with reference to the “B”arrows, a second material, such as an assay sample, can flow throughsecond channel 110, past second isolation valve 112, through via 111,and into second chamber 120. To allow flow into the reaction chambers140, 120, first and second isolation valves 132, 112, respectively, areboth in an open valve state. To prevent or inhibit flow between firstreaction chamber 140 and second reaction chamber 120 through reactionchannel 170, interface valve 162 is in a closed valve state. Under suchconditions, first channel 130 is in open fluid communication with firstreaction chamber 140, and second channel 110 is in fluid communicationwith second reaction chamber 120, whereas fluid communication betweenthe first and second chambers is interrupted or inhibited. Reactionchamber sizes may vary. In some embodiments, the volume of secondreaction chamber 120 is different or greater than the volume of firstreaction chamber 140. For example, the volume of second reaction chamber120 can be ten times greater than the volume of first reaction chamber140. Materials can be loaded into their respective chambers underpressure. Relatedly, materials can be loaded into chambers at certainconcentrations. In some cases, a reagent solution is loaded into achamber at a 10× concentration, and is then diluted when reacted with asample solution contained in another chamber.

After the first and second materials have been loaded into first andsecond reaction chambers 140, 120 respectively, the control channel 150can be activated so as to transform each of first and second isolationvalves 132, 112 from an open valve state to a closed valve state. Inthis way the materials can be confined, optionally under pressure,within the reaction chambers. Hence, it is understood that a singlecontrol channel, for example control channel 150, can control flow of afirst material into a first reaction chamber, and can also control flowof a second material into a second reaction chamber. Operation of asingle control channel can thus act to isolate a first volume ofmaterial or solution within the first chamber via actuation of the firstisolation valve 132, and can also isolate a second volume of material orsolution within the second chamber via actuation of the second isolationvalve 112. Relatedly, operation of a single control channel can cause afirst deflection in a first direction at first isolation valve 132, anda second deflection in a second direction at second isolation valve 112,where first direction is opposite to second direction. For example, thedeflection in the first isolation valve can be in the upward direction,and the deflection in the second isolation valve can be in the downwarddirection. Accordingly, control of more than one isolation valve can beeffected simultaneously by operation of the single control channel.Materials can be confined within the reaction chambers under anysuitable amount of pressure. In some embodiments, the pressure in thefirst reaction chamber 140 is different or greater than the pressure inthe second reaction chamber 120. For example, a first material such as areagent can be disposed in first reaction chamber 140 at a firstpressure that is within a range from about 0 psi to about 15 psi.Relatedly, a second material such as a sample can be disposed in secondreaction chamber 120 at a second pressure that is within a range fromabout 0 psi to about 10 psi. In some instances, material can becontained in the first reaction chamber 140 at about 10 psi, andmaterial can be contained in the second reaction chamber 120 at about 0psi. Often, loading of the microfluidic device involves introducingmaterial into first channel 130 or second channel 110, or both, underpressure. A pressurizing mechanism can be used to drive materials intothe chambers.

In some cases, embodiments are directed to systems and methods forconducting one or more reactions at one or more selected temperatures orranges of temperatures over time. A microfluidic system may include aplurality of separate reaction chambers formed in a multi-layerelastomeric block. The system may also include a thermal transfer deviceproximal to or near at least one of the reaction chambers. The thermaltransfer device can be formed to contact a thermal control source.Reagents for carrying out a desired reaction can be introduced into amicrofluidic array device or matrix. The array device or matrix can becontacted with the thermal control device such that the thermal controldevice is in thermal communication with the thermal control source sothat a temperature of the reaction in at least one of the reactionchamber is changed or controlled as a result of a change in temperatureof the thermal control source. Exemplary thermal cycling techniques arediscussed in U.S. Patent Publication No. 2007/0196912, the content ofwhich is incorporated herein by reference. In some embodiments, amicrofluidic device or chip can be coupled with or in operativeassociation with an Integrated Heat Spreader (IHS). Such heatingmechanisms are discussed in U.S. Pat. No. 7,307,802, the content ofwhich is incorporated herein by reference.

FIG. 1A shows an exploded perspective view of individual layers of aunit cell 100 of a microfluidic device, according to embodiments of thepresent invention. Each layer typically includes an elastomeric membranewith one or more recesses, channels, chambers, or the like. As depictedhere, first layer 181 of unit cell 100 includes a first channel 130 influid communication with a first chamber 140. First layer 181 alsoincludes a second chamber 120. Second layer 182 includes a controlchannel 150, a first via 111, and a second via 131. Third layer 183includes a second channel 110 and a reaction channel 170. As furtherdiscussed elsewhere herein, unit cell 100 can be configured so thatsecond channel 110 and reaction channel 170 are in fluid communicationwith second chamber 120, optionally by way of via 111. For example,creating a fluid passage that extends from second channel 110 toreaction channel 170 can involve removing a portion of second layer 182that is disposed below second chamber 120. Creation of this fluidpassage can also involve removing a corresponding portion of third layer183 that is disposed below second chamber 120. Similarly, unit cell 100can be configured so that reaction channel 170 is in fluid communicationwith first chamber 140, optionally by way of via 131. For example,creating a fluid passage that extends from first channel 130 to reactionchannel 170 can involve removing a portion of second layer 182 that isdisposed below first chamber 140. Creation of this fluid passage canalso involve removing a corresponding portion of third layer 183 that isdisposed below first chamber 140. Fourth layer 184 includes an interfacechannel 160.

Hence, as shown here, first channel 130 is at least partially containedwithin a first layer 181. Control channel 150, via 111, and via 131 areeach at least partially contained within a second layer 182, where thesecond layer is adjacent to the first layer. Second channel 110 andreaction channel 170 are at least partially contained within a thirdlayer 183, where the third layer is adjacent to the second layer. Asshown here, vias 131, 111 are disposed in second layer 182. It isunderstood that corresponding vias can be formed in third layer 183, soas to provide fluid communication from chamber 140 through via 131 andinto channel 170, and fluid communication from chamber 120 through via111 and into the intersection of channels 110 and 170. Interface channel160 is at least partially contained within a fourth layer 184, where thefourth layer is adjacent to the third layer. First chamber 140 is atleast partially contained within first layer 181. First chamber 140 insome instances can also be least partially contained within or incommunication with passages located in second layer 182 and third layer183, thus providing fluid communication between first chamber 140 andfirst channel 130, and between first chamber 140 and reaction channel170. Second chamber 120 is at least partially contained within firstlayer 181. In some instances second chamber 120 can be at leastpartially contained within or in communication with passages located insecond layer 182 and third layer 183, thus providing fluid communicationbetween second chamber 120 and reaction channel 170.

According to the embodiment shown in FIG. 1, reaction channel 170 andinterface valve 162 are not located within the same plane or level asfirst chamber 140 and second chamber 120. For example, reaction channel170 is disposed in third layer 183, interface valve 162 operates at ornear the boundary or junction between fourth layer 184 and third layer183, and first and second chambers 140, 120 are disposed in first layer181. As the routing passage or reaction channel 170 passes in a lower ordifferent layer than that of the chambers, this allows the chambers tobe located in close proximity with one another. In some embodiments, asidewall of first chamber 140 and a facing sidewall of second chamber120 are separated by a distance of about 120 microns. In relatedembodiments, a distance between facing sidewalls of first and secondchambers is within a range from about 40 microns to about 225 microns.For example, a first chamber sidewall and a facing second chambersidewall can be separated by a distance of about 50 microns, about 60microns, about 70 microns, or about 80 microns. Often, interface valve162 is about 50 microns in width. Hence, the distance between facingsidewalls of first and second chambers can be less than, about the sameas, or more than the diameter or width of the interface valve whichcontrols or modulates flow between the chambers. Accordingly,microfluidic devices employing such architecture can present extremelylarge numbers of chambers within a given area. Such high densities maybe difficult to achieve in situations where a valve that controls flowbetween two chambers is disposed in the same layer as the chambers.

First chamber 140 can have a width within a range from about 25 micronsto about 75 microns, a length within a range from about 80 microns toabout 240 microns, and a height within a range from about 30 microns toabout 90 microns. Relatedly, first chamber 140 can have a volume withina range from about 0.1 nanoliters to about 10 nanoliters. For example,first chamber 140 can have a width of 50 microns, a length of 162.5microns, a height of 60 microns, and a volume of 0.49 nanoliters. Secondchamber 120 can have a width within a range from about 70 microns toabout 210 microns, a length within a range from about 80 microns toabout 240 microns, and a height within a range from about 150 microns toabout 450 microns. Relatedly, second chamber 120 can have a volumewithin a range from about 1 nanoliter to about 20 nanoliters. Forexample, second chamber 120 can have a width of 137.5 microns, a lengthof 162.5 microns, a height of 300 microns, and a volume of 6.7nanoliters. A microfluidic device according to embodiments of thepresent invention can provide a center-to-center distance between firstchamber 120 and second chamber of about 300 microns. In some cases, thiscenter-to-center distance is within a range from about 250 microns toabout 350 microns. Optionally, the center-to-center distance between thefirst chamber and the second chamber is about 312.5 microns.

In some embodiments, a microfluidic device can include one or morelayers that have been prepared according to spin or pour fabricationprotocols. For example, a spin protocol can involve placing a polymericmaterial on a patterned disc or mold, and spinning the disc to create alayer of polymer across the disc. Exemplary polymers includepolymethylmethacrylate, polystyrene, polypropylene, polyester,fluoropolymers, polytetrafluoroethylene, polycarbonate, polysilicon, andpolydimethylsiloxane (PDMS). A pour protocol can involve pouring a PDMSmaterial, for example, on a patterned template or mold, which can resultin a layer of PDMS which can be peeled or pulled off the mold intact.Often, a layer prepared by a pour fabrication technique is thicker thana layer prepared by a spin fabrication technique. Elastomeric blocks caninclude one or more pour or spin layers, in any desired combination. Insome embodiments, first layer 181 can be fabricated according to a pourprotocol. For example, PDMS can be poured onto a mold that has raisedportions corresponding to the various desired fluid flow channels andchambers. FIG. 1B shows an exemplary mold 190 b which can be used tofabricate first layer 181 of FIG. 1A. After curing, the first layer canbe peeled away from the mold. First layer 181 can include openings,recesses, or other voids that at least partially form or define firstchannel 130, first chamber 140, and second chamber 120. To create secondlayer 182, PDMS can be placed onto a mold that has raised portionscorresponding to the various desired containment or control channels.FIG. 1C shows an exemplary mold 190 c which can be used to fabricatesecond layer 182 of FIG. 1A. Mold 190 c can also include, for example,raised or contoured portions 191 c that form corresponding marks insecond layer 182. These marks can be used during a laser ablationprocedure, such that the laser ablation is directed toward the marksduring the ablation. Mold 190 c can be spun, so as to provide a thinlayer of PDMS across the mold. Second layer 182 can include openings,recesses, or other voids that at least partially form or define controlchannel 150. In some cases, second layer 182 can be exposed to one ormore laser ablations. An ablative laser beam directed to second layer182 can form vias 111, 131. After second layer 182 is sufficientlycured, first layer 181 can be aligned and contacted with the secondlayer. The first layer can adhere with the second layer, and both layerscan be peeled off mold 190 c simultaneously. To create third layer 183,PDMS can be placed onto a mold that has raised portions corresponding tothe various desired containment or control channels. FIG. 1D shows anexemplary mold 190 d which can be used to fabricate third layer 183 ofFIG. 1A. The mold can be spun, so as to provide a thin layer of PDMSacross the mold. Third layer 183 can include openings, recesses, orother voids that at least partially form or define second channel 110and reaction channel 170. After third layer 183 is sufficiently cured,the combined first layer 181 and second layer 182 can be aligned andcontacted with the third layer. The third layer can adhere with thesecond layer, and all three layers can be peeled off mold 190 dsimultaneously. To create fourth layer 184, PDMS can be placed onto amold that has raised portions corresponding to the various desiredcontainment or control channels. FIG. 1E shows an exemplary mold 190 ewhich can be used to fabricate fourth layer 184 of FIG. 1A. The mold canbe spun, so as to provide a thin layer of PDMS across the mold. Fourthlayer 184 can include openings, recesses, or other voids that at leastpartially form or define interface channel 160. After fourth layer 184is sufficiently cured, the combined first layer 181, second layer 182,and third layer 183 can be aligned and contacted with the fourth layer.The fourth layer can adhere with the third layer, and all four layerscan be peeled off mold 190 e simultaneously. Optionally, the four layerscan be placed on or contacted with a fifth layer 186 as shown in FIG.1A. The fifth layer can include a laminate or tape, or a similarlysuitable material, which operates to seal a recess in the fourth layer,so as to form or seal interface channel 160. In this way, the combinedfirst, second, third, and fourth layers can then be placed on orcontacted with the fifth layer, which may be a solid spin layer. Thefifth layer can act as a sealing layer. According to some embodiments,the fifth layer may include an elastomeric material, such as PDMS. Insome cases, the fifth layer can include a rigid or hard material such asglass, silicon, or a plastic such as polystyrene. The fifth layer may,for example, seal recesses formed in bottom of the fourth layer, so asto provide channels in the fourth layer. The sealing layer can include afilm which may be attached to the fourth layer via an adhesive. Hence,the sealing layer can form channels from recesses molded or machinedinto an adjacent layer. The sealing layer can be a transparent material,for example, polystyrene, polycarbonate, or polypropylene. Relatedly,the sealing layer can be flexible, such as an adhesive tape, and may besuitable for attachment to a substrate by bonding, such as with adhesiveor heat sealing, or mechanically attached such as by compression. Often,materials used to fabricate a sealing layer are compliant to formfluidic seals with each recess to form a fluidic channel with minimalleakage. A sealing layer may further be supported by an additionalsupport layer that is rigid (not shown). In some cases, a sealing layeris rigid.

In some cases, passages or vias can be formed between channels orchambers at one layer and channels or chambers at another layer. Forexample, it is possible to create a via 131 through second layer 182 toprovide fluid communication between first chamber 140 in first layer 181and reaction channel 170 in third layer 183. Similarly, it is possibleto create a via 111 through second layer 182 to provide fluidcommunication between second chamber 120 in first layer 181 and secondchannel 110 and reaction channel 170 in third layer 183. In someinstances, creation of these vias can enlarge the volume of the reactionchambers. In some cases, the vias can be formed by using a laser punchto remove or ablate portions of elastomeric membrane. As shown in FIG.1A, for example, reaction chambers 120, 140 can have an interior spacethat extends above a plane defined by the top of first channel 130. Thisinterior space can also extend above a plane defined by the top ofsecond channel 110, and above a plane defined by the top of reactionchannel 170. Hence, during loading of the unit cell, fluid can flowthrough first channel 130 and upward into the interior of first chamber140. Similarly, during loading, fluid can flow through second channel110 and upward into the interior of second chamber 120, optionallythrough a via formed in the second layer. Relatedly, during a mixingoperation, fluid can flow from the interior of first chamber 140 anddownward into reaction channel 170, optionally through a via 131 formedin the second layer. Similarly, during a mixing operation fluid can flowfrom reaction channel 170 and upward into the interior of second chamber120, optionally through a via 111 formed in the second layer.

According to embodiments of the present invention, the second, third,fourth, and fifth layers can be processed or laser punched as part of aprocedure that forms a loading passage to first channel 130 in firstlayer 181. Relatedly, the fourth and fifth layers can be processed orlaser punched as part of a procedure that forms a loading passage tosecond channel 110 in third layer 183. Loading passages, vias, and thelike can be formed using a drilling or ablation mechanism. For example,a loading passage or via can be fabricated by ablating a portion of theelastomeric block. Excimer lasers are well suited for such ablationtechniques, as they can produce a laser beam which removes a portion ofthe elastomeric block. In some cases, loading passages or vias, orportions thereof, can be formed before one or more of the individuallayers are adhered together. For example, a portion of a loading passageor via can be formed in a layer during the molding process, or after themolding process and before the adhesion process. Optionally, formationof at least a portion of the loading passage or via can involve etchingone or more elastomeric layers prior to forming the complete multilayerelastomeric block.

Accordingly, in some embodiments fabrication methods include forming afirst elastomeric layer on top of a first micromachined mold, where thefirst micromachined mold has one or more raised protrusions that formone or more recesses along a bottom surface of the first elastomericlayer. Methods may also include forming a second elastomeric layer ontop of a second micromachined mold, where the second micromachined moldhas one or more raised protrusions that form one or more recesses alonga bottom surface of the second elastomeric layer. Methods may includebonding or adhering the bottom surface of the first elastomeric layeronto a top surface of the second elastomeric layer such that a channelor chamber forms in a recess between the first and second elastomericlayers. Any desired number of layers can be fabricated in this way.Methods also include positioning the last or final elastomeric layer ontop of a planar substrate such that a channel or chamber forms in arecess between the final elastomeric layer and the planar substrate.Embodiments of the present invention encompass multilayer microfluidicdevices having any desired elastomeric valve or pump configuration whichincludes such channels or chambers. Exemplary elastomeric valve and pumpfabrication techniques are described in U.S. Pat. No. 6,408,878, thecontent of which is incorporated herein by reference.

Microfluidic device embodiments of the invention can be constructed outof any material or combination of materials that can be fabricated tohave microfluidic channels and chambers, and valves that regulate flowthrough channels and into chambers. Materials from which a device can befabricated include, without limitation, elastomers, silicon, glass,metal, polymer, ceramic, inorganic materials, and/or combinations ofthese materials.

The methods used in fabrication of a microfluidic device may vary withthe materials used, and include soft lithography methods, microassembly,bulk micromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,modular assembly methods, replica molding methods, injection moldingmethods, hot molding methods, laser ablation methods, combinations ofmethods, and other methods known in the art or developed in the future.A variety of exemplary fabrication methods are described in Fiorini andChiu, 2005, “Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; U.S.Pat. No. 6,767,706 B2, e.g., Section 6.8 “Microfabrication of a SiliconDevice”; Terry et al., 1979, A Gas Chromatography Air AnalyzerFabricated on a Silicon Wafer, IEEE Trans. on Electron Devices, v.ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis Systems,New York, Kluwer; Webster et al., 1996, Monolithic Capillary GelElectrophoresis Stage with On-Chip Detector in International ConferenceOn Micro Electromechanical Systems, MEMS 96, pp. 491496; and Mastrangeloet al., 1989, Vacuum-Sealed Silicon Micromachined Incandescent LightSource, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506. Each ofthese references are incorporated herein by reference for all purposes.

In preferred embodiments, the device is fabricated using elastomericmaterials. Fabrication methods using elastomeric materials and methodsfor design of devices and their components have been described in detailin the scientific and patent literature. See, e.g., Unger et al., 2000,Science 288:113-16; U.S. Pat. Nos. 6,960,437 (Nucleic acid amplificationutilizing microfluidic devices); 6,899,137 (Microfabricated elastomericvalve and pump systems); 6,767,706 (Integrated active flux microfluidicdevices and methods); 6,752,922 (Microfluidic chromatography); 6,408,878(Microfabricated elastomeric valve and pump systems); 6,645,432(Microfluidic systems including three-dimensionally arrayed channelnetworks); U.S. Patent Application publication Nos. 2004/0115838,2005/0072946; 2005/0000900; 2002/0127736; 2002/0109114; 2004/0115838;2003/0138829; 2002/0164816; 2002/0127736; and 2002/0109114; PCT patentpublications WO 2005/084191; WO 05030822A2; and WO 01/01025; Quake &Scherer, 2000, “From micro to nanofabrication with soft materials”Science 290: 1536-40; Xia et al., 1998, “Soft lithography” AngewandteChemie-International Edition 37:551-575; Unger et al., 2000, “Monolithicmicrofabricated valves and pumps by multilayer soft lithography” Science288:113-116; Thorsen et al., 2002, “Microfluidic large-scaleintegration” Science 298:580-584; Chou et al., 2000, “MicrofabricatedRotary Pump” Biomedical Microdevices 3:323-330; Liu et al., 2003,“Solving the “world-to-chip” interface problem with a microfluidicmatrix” Analytical Chemistry 75, 4718-23,” Hong et al, 2004, “Ananoliter-scale nucleic acid processor with parallel architecture”Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005, “Disposablemicrofluidic devices: fabrication, function, and application”Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidic tectonics: acomprehensive construction platform for microfluidic systems.” Proc.Natl. Acad. Sci. USA 97:13488-13493; Rolland et al., 2004,“Solvent-resistant photocurable “liquid Teflon” for microfluidic devicefabrication” J. Amer. Chem. Soc. 126:2322-2323; Rossier et al., 2002,“Plasma etched polymer microelectrochemical systems” Lab Chip 2:145-150;Becker et al., 2002, “Polymer microfluidic devices” Talanta 56:267-287;Becker et al., 2000, and other references cited herein and found in thescientific and patent literature. Each of these references areincorporated herein by reference for all purposes.

Embodiments of the present invention further encompass aspects ofmicrofluidic fabrication and production, as well as microfluidic deviceoperation and use, as disclosed in U.S. patent application Ser. No.12/018,138 filed Jan. 22, 2008, the content of which is incorporatedherein by reference for all purposes.

Any of a variety of ablation, etching, or similar techniques can be usedto form vias or passages in an elastomeric block, membrane, or layer.Such etching procedures are well suited for creating elastomeric layershaving multiple holes or apertures, for example. In an exemplaryprocess, an elastomeric material is placed on a wafer or mold, andallowed to cure. The elastomeric material can include one or morepolymers incorporating materials such as chlorosilanes or methyl-,ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) such as DowChemical Corp. Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical may also be used. In some cases, the elastomeric material isdeposited on the wafer or mold in a spin coating process, a spraycoating process, a dip coating process, a screen printing process, aninkjet deposition process, or the like. The curing procedure can involvebaking or room temperature vulcanizing (RTV), photocuring, and the like.

An elastomeric composition may include multiple parts, which can bemixed together at various ratios to obtain desired bond properties. Forexample, an elastomeric material may include a Part A and a Part B,which when mixed together in prescribed amounts facilitates the desiredbond parameters. In some cases, the parts may be mixed in a ratio withina range from about 3:1 to about 30:1. For example, an elastomeric PDMScomposition is baked to provide a 10:1 RTV layer.

In some cases, a photoresist material can be placed on the curedelastomeric material. For example, an SU-8 resist (available fromMicroChem Corp., Newton Mass.) can be applied to the elastomer.Exemplary SU-8 resists include SU-8 2000, SU-8 3000, SU-8 2007, SU-83005, and the like. The photoresist can be deposited on the elastomericmaterial in a spin coating procedure, at a desired rotational speed andduration. In some cases, the spin coating can be performed at arotational speed within a range from about 1000 to about 10,000 rpm, andfor a duration within a range from about 20 seconds to about 2,000seconds. For example, the spin coating can be performed at 5000 rpm for200 seconds. Following this deposition, the photoresist mask can have athickness or depth within a range from about 0.5 to about 50 microns. Insome cases, the thickness is about 5 microns. The thickness of the maskcan be selected for facile via opening formation, and the selected spintime can eliminate or inhibit beading of the photoresist material. Thephotoresist material can be used as an etch mask.

Additional procedures can be performed to prepare the photoresist forlithography exposure. For example, the photoresist can be processed at aselected temperature for a selected time duration. In some embodiments,the photoresist is soft baked at a temperature within a range from about45° C. to about 85° C. Relatedly, the photoresist can be baked for aduration within a range from about 1 minute to about 10 minutes. In somecases, the soft bake is performed for 5 minutes at 65° C. Suchpreparation techniques can help to eliminate or inhibit photoresist maskcracking at exposure. The preparation procedure may also include coolingthe photoresist. For example, the photoresist may be cooled at roomtemperature or at a temperature within a range from about 18° C. toabout 37° C., and for a duration within a range from about 3 minutes toabout 300 minutes. In some cases, the photoresist is cooled for about 30minutes.

The lithography procedure can involve multiple exposure steps. Forexample, a first exposure step can be performed with a first exposuremask, and a second subsequent exposure step can be performed with asecond exposure mask. An exposure step can involve the application ofradiation or energy, through an exposure mask, toward a photoresist.Exposure radiation can include ultraviolet light, near ultravioletlight, deep ultraviolet light, visible light, infrared light, or energyat any desired wavelength along the electromagnetic spectrum. In somecases, exposure radiation is delivered at one or more wavelengths withina range from about 10 to about 10⁻⁹ cm. In some cases, the type ofradiation or energy is selected based on the composition of thephotoresist. For example, specific types of radiation or energy can beapplied to I-line photoresists, G-line photoresists, H-linephotoresists, and the like.

The use of multiple masks can help to prevent or inhibit the effect ofcontaminants on a mask from replicating on the photoresist. For example,if there is an unwanted particle on the first mask at a certainlocation, exposure with a second mask can help to ensure exposure of thephotoresist at that location. The exposure process can be followed witha post-exposure bake (PEB) procedure. In some cases, a PEB procedure isperformed for a duration within a range from about 0 to about 200minutes, and at a temperature within a range from about 50° C. to about80° C. For example, a PEB can be performed for 2 minutes at 65° C. Insome cases, the PEB can operate to cross-link the photoresist maskmaterial, rendering the material nonsoluble. Thereafter, the exposedphotoresist can be allowed to cool. An exemplary cooling process isperformed at a temperature within a range from about 18° C. to about 37°C. for a duration within a range from about 1 hour to about 40 hours. Insome cases, the exposed photoresist is cooled at room temperature forabout 18 hours.

A development process can be performed following exposure. In somecases, the photoresist mask is developed for a duration within a rangefrom about 10 seconds to about 10 minutes. During the developmentprocess a developer is applied to the exposed photoresist. The developercan include, for example, an organic solvent such as acetate. It isunderstood that the developer or solvent may be selected based on thecomposition of the photoresist. The developer can operate to dissolve ordegrade areas or locations of the photoresist layer that were unexposedor masked during the exposure process. Following development, thephotoresist mask is subject to a drying procedure. For example, the maskcan be spin-dried. The mask may also be allowed to relax for a desiredperiod of time. In some cases, the mask is allowed to relax at or nearroom temperature for a duration within a range from about 1 minute toabout 48 hours.

Optionally, additional elastomeric layers can be spin-coated orotherwise applied onto the developed photoresist. For example, an RTVcoating have a thickness or depth within a range from about 0.3 micronsto about 30 microns can be deposited on the photoresist mask. Theelastomeric coating can be baked for a duration within a range fromabout 5 minutes to about 3 hours, at a temperature within a range fromabout 40° C. to about 80° C. In some cases, a 3 micron RTV coating isspin-coated on an SU-8 mask, and baked for 1 hour at 60° C. Suchtechniques can help to minimize lateral etch, and reduce via sizenon-uniformity. The RTV layer can be deposited on a patternedphotoresist layer, to help prevent or inhibit the formation of pinholesin the underlying elastomer.

Typically, etching involves removing certain areas of the elastomericmaterial that are not protected by the photoresist followingdevelopment. In an exemplary procedure, etching can be performed for aduration within a range from about 1 minute to about 20 minutes and at atemperature within a range from about 50° C. to about 90° C. Forexample, etching can be carried out in an 80% tetrabutylammoniumfluoride (TBAF) etchant solution for 6-8 minutes at 70 degrees ° C. Insome cases, etching is performed in an ultrasonic bath tank, optionallyin degas mode. Such procedures can help to ensure a uniform etchingdepth, with minimum damage to a photoresist mask during the etchingprocedure. Etching can be followed with a deionized water wash. In somecases, a hot water wash is performed for three minutes. The photoresistmask can be removed with adhesive tape. A deionized water wash can beapplied again, optionally for 3 minutes. Stacking procedures can provideadditional layers to the elastomer. In some cases, adjacent layersadhere to one another by way of interlayer bonding.

FIG. 2 illustrates another perspective view of unit cell 100. As shownhere with reference to the “A” arrows, the first material can flow fromfirst reaction chamber 140, through via 131, through reaction channel170, past interface valve 162, through via 111, and into second reactionchamber 120, where the first material can contact the second material.It is understood that in some embodiments, the second material can flowfrom second reaction chamber 120, through via 111, through reactionchannel 170, past interface valve 162, through via 131, and into firstreaction chamber. To allow such flow through reaction channel 170, theinterface channel 160 can be activated so as to transform interfacevalve 162 from a closed valve state to an open valve state. Under suchconditions, the first and second reaction chambers 140, 120 are in openfluid communication by way of the reaction channel and the vias. When,for example, material contained in first reaction chamber 140 is morehighly pressurized relative to material contained in second reactionchamber 120, the pressure differential can help to release or openinterface valve 162. Relatedly, such a pressure differential canfacilitate mixing between the first material and the second material, asthe first material is forcefully expelled from first chamber and intosecond chamber, thus squirting a stream of first material into a secondmaterial contained in the second chamber, where the first material candiffuse into or permeate through the second material. Often, thepresence, absence, or extent of any reaction between the first andsecond materials, or involving either or both of the first and secondmaterials, within the second reaction chamber can be characterized,confirmed, detected, or quantified by inspection, for example with areader, sensor, or imaging device 190. An imaging device 190 can includea camera, optionally having a charge-coupled device (CCD), that detectsor monitors energy that emits from the chamber. In some cases, theimaging device can detect emission intensity output. Exemplary imagingdevices and reader techniques suitable for use with embodiments of thepresent invention are described in U.S. Pat. No. 7,307,802 issued Dec.11, 2007, the content of which is incorporated herein by reference. Insome cases, a reaction within a chamber is facilitated by a thermalcycler. Embodiments of the present invention encompass systems andmethods for mixing or reacting materials within chambers, where suchmixing or reacting procedures involve any of a variety of desiredthermal cycling heating protocols or thermal gradient modalities.

FIG. 3 illustrates a perspective view of a matrix 300 having four unitcells 300 _((1,1)), 300 _((1,2)), 300 _((2,1)), and 300 _((2,2))arranged in two rows and two columns. Matrix 300 includes a plurality offirst channels 330 ₍₁₎, 330 ₍₂₎, a plurality of first isolation valves332 _((1,1)), 332 _((1,2)), 332 _((2,1)), 332 _((2,2)), a plurality offirst chambers 340 _((1,1)), 340 _((1,2)), 340 _((2,1)), 340 _((2,2)), aplurality of second channels 310 ₍₁₎, 310 ₍₂₎, a plurality of secondisolation valves 312 _((1,1)), 312 _((1,2)), 312 _((2,1)), 312 _((2,2)),a plurality of second chambers 320 _((1,1)), 320 _((1,2)), 320 _((2,1)),320 _((2,2)), a plurality of control channels 350 ₍₁₎, 350 ₍₂₎, aplurality of interface channels 360 ₍₁₎, 360 ₍₂₎, and a plurality ofreaction channels 370 _((1,1)), 370 _((1,2)), 370 _((2,1)), 370_((2,2)). It is appreciated that the unit cell architecture embodimentsdisclosed herein can be scaled to provide a matrix having any number ofdesired unit cells. For example, a matrix can include 9216 unit cellsarranged in 96 rows and 96 columns. Hence, embodiments of the presentinvention provide a high density format for reacting a plurality ofsamples with a plurality of reagents, for example, ninety-six (96)samples with ninety-six (96) reagents.

Microfluidic device features such as channels, valves, chambers, areoften at least partially contained, embedded, or formed by or within oneor more layers of an elastomeric block 380. As shown here with referenceto the “A1” arrows, a first material such as a reagent can flow througha first channel 330 ₍₁₎, past or through a plurality of first isolationvalves 332 _((1,1)), 332 _((1,2)), and into a plurality of firstchambers 340 _((1,1)), 340 _((1,2)), respectively. Likewise, withreference to the “A2” arrows, a second material such as a reagent canflow through a first channel 330 ₍₂₎, past or through a plurality offirst isolation valves 332 _((2,1)), 332 _((2,2)), and into a pluralityof first chambers 340 _((2,1)), 340 _((2,2)), respectively. In someembodiments, materials flow through first channel 330 ₍₁₎ and firstchannel 330 ₍₂₎ in the same direction. In some embodiments, materialflowing through first channel 330 ₍₁₎ travels in a direction oppositefrom material flowing through first channel 330 ₍₂₎. With reference tothe “B1” arrows, a third material such as a sample can flow through asecond channel 310 ₍₁₎, past or through a plurality of second isolationvalves 312 _((1,1)), 312 _((2,1)), and into a plurality of secondchambers 320 _((1,1)), 320 _((2,1)), respectively. Hence, embodiments ofthe present invention provide microfluidic techniques whereby a materialcan be flowed through a common passage or trunk of a channel, such assecond channel 310 ₍₁₎, and into a plurality of individual branchesstemming from the common trunk, such as those branch channels whichindividually feed into second chambers 320 _((1,2)), 320 _((2,2)).Similarly, with reference to the “B2” arrows, a fourth material such asa sample can flow through a second channel 310 ₍₂₎, past or through aplurality of second isolation valves 312 _((1,2)), 312 _((2,2)), andinto a plurality of second chambers 320 _((1,2)), 320 _((2,2)),respectively. In some embodiments, materials flow through second channel310 ₍₁₎ and second channel 310 ₍₂₎ in the same direction. In someembodiments, material flowing through second channel 310 ₍₁₎ travels ina direction opposite from material flowing through second channel 310₍₂₎. As shown in FIG. 3, material flowing through second channel 310 ₍₁₎and material flowing through second channel 310 ₍₂₎ travel in opposingdirections “B1” and “B2”, respectively. Hence, when loading multiplesamples into an elastomeric layered block, some samples can beintroduced through routing lines on one side of the block, and somesamples can be introduced through routing lines on an opposing side ofthe block. This allows for an even distribution or placement of sampleloading route lines on opposite sides of the block, instead of placingall or most sample loading route lines on the same side of the block.Because the sum total of the sample routing lines are divided betweendifferent sides of the block, more sample routing lines can beintroduced into the block. Consequently, a greater number of samples canbe analyzed within the block during a single procedure.

To allow flow from the plurality of first channels 330 ₍₁₎, 330 ₍₂₎ intothe plurality of first reaction chambers 340 _((1,1)), 340 _((1,2)), 340_((2,1)), 340 _((2,2)), each of the plurality of first isolation valves332 _((1,1)), 332 _((1,2)), 332 _((2,1)), 332 _((2,2)) is in an openvalve state. To allow flow from the plurality of second channels 310₍₁₎, 310 ₍₂₎ into the plurality of second reaction chambers 320_((1,1)), 320 _((1,2)), 320 _((2,1)), 320 _((2,2)), each of theplurality of second isolation valves 312 _((1,1)), 312 _((1,2)), 312_((2,1)), 312 _((2,2)) is in an open valve state. To prevent or inhibitflow between each of the plurality of first reaction chambers 340_((1,1)), 340 _((1,2)), 340 _((2,1)), 340 _((2,2)) and theircorresponding counterpart of the plurality of second reaction chambers320 _((1,1)), 320 _((1,2)), 320 _((2,1)), 320 _((2,2)) through theircorresponding counterpart of the plurality of reaction channels 370_((1,1)), 370 _((1,2)), 370 _((2,1)), 370 _((2,2)), respectively, eachof the plurality of interface valves 362 _((1,1)), 362 _((1,2)), 362_((2,1)), 362 _((2,2)), respectively, is in a closed valve state. Undersuch conditions, first channel 330 ₍₁₎ is in fluid communication withfirst reaction chambers 340 _((1,1)), 340 _((1,2)), first channel 330₍₂₎ is in fluid communication with first reaction chambers 340 _((2,1)),340 _((2,2)), second channel 310 ₍₁₎ is in fluid communication withsecond reaction chambers 320 _((1,1)), 320 _((2,1)), and second channel310 ₍₂₎ is in fluid communication with second reaction chambers 320_((1,2)), 320 _((2,2)). Fluid communication between first chambers 340_((1,1)), 340 _((1,2)), 340 _((2,1)), 340 _((2,2)) and second chambers320 _((1,1)), 320 _((1,2)), 320 _((2,1)), 320 _((2,2)), respectively, isinterrupted.

A first material can be loaded into first reaction chambers 340_((1,1)), 340 _((1,2)) via first channel 330 ₍₁₎. A second material canbe loaded into first reaction chambers 340 _((2,1)), 340 _((2,2)) viafirst channel 330 ₍₂₎. A third material can be loaded into secondreaction chambers 320 _((1,1)), 320 _((2,1)) via second channel 310 ₍₁₎.A fourth material can be loaded into second reaction chambers 320_((1,2)), 320 _((2,2)) via second channel 310 ₍₂₎. Optionally, suchmaterials can be loaded into the chambers under a desired or selectedpressure. Control channel 350 ₍₁₎ can be activated so as to transformeach of first isolation valves 332 _((1,1)), 332 _((2,1)) and secondisolation valves 312 _((1,1)), 312 _((2,1)) from an open valve state toa closed valve state. Similarly, control channel 350 ₍₂₎ can beactivated so as to transform each of first isolation valves 332_((1,2)), 332 _((2,2)) and second isolation valves 312 _((1,2)), 312_((2,2)) from an open valve state to a closed valve state. In this waythe materials can be confined or maintained, optionally under pressure,within the reaction chambers.

FIG. 4 illustrates another perspective view of matrix 300 having fourunit cells 300 _((1,1)), 300 _((1,2)), 300 _((2,1)), 300 _((2,2)). Asshown here with reference to the “A” arrows, the first material can flowfrom first reaction chamber 340 _((1,1)), through reaction channel 370_((1,1)), past interface valve 362 _((1,1)), and into second reactionchamber 320 _((1,1)), where the first material can contact the thirdmaterial. To allow such flow through reaction channel 370 _((1,1)), theinterface channel 360 ₍₁₎ can be activated so as to transform interfacevalve 362 _((1,1)) from a closed valve state to an open valve state.Under such conditions, first reaction chamber 340 _((1,1)) and secondreaction chamber 320 _((1,1)) are in fluid communication via reactionchannel 370 _((1,1)). Often, the presence, absence, or extent of anyreaction between the first and third materials within second reactionchamber 320 _((1,1)) can be confirmed, detected, or quantified byinspection, for example with a reader or sensor 390.

With reference to the “B” arrows, the first material can flow from firstreaction chamber 340 _((1,2)), through reaction channel 370 _((1,2)),past interface valve 362 _((1,2)), and into second reaction chamber 320_((1,2)), where the first material can contact the fourth material. Toallow such flow through reaction channel 370 _((1,2)), the interfacechannel 360 ₍₁₎ can be activated so as to transform interface valve 362_((1,2)) from a closed valve state to an open valve state. Under suchconditions, first reaction chamber 340 _((1,2)) and second reactionchamber 320 _((1,2)) are in fluid communication via reaction channel 370_((1,2)). Often, the presence, absence, or extent of any reactionbetween the first and third materials within second reaction chamber 340_((1,2)) can be confirmed, detected, or quantified by inspection, forexample with a reader or sensor 390.

With reference to the “C” arrows, the second material can flow fromfirst reaction chamber 340 _((2,1)), through reaction channel 370_((2,1)), past interface valve 362 _((2,1)), and into second reactionchamber 320 _((2,1)), where the second material can contact the thirdmaterial. To allow such flow through reaction channel 370 _((2,1)), theinterface channel 360 ₍₂₎ can be activated so as to transform interfacevalve 362 _((2,1)) from a closed valve state to an open valve state.Under such conditions, first reaction chamber 340 _((2,1)) and secondreaction chamber 320 _((2,1)) are in fluid communication via reactionchannel 370 _((2,1)). Often, the presence, absence, or extent of anyreaction between the first and third materials within second reactionchamber 320 _((2,1)) can be confirmed, detected, or quantified byinspection, for example with a reader or sensor 390.

With reference to the “D” arrows, the second material can flow fromfirst reaction chamber 340 _((2,2)), through reaction channel 370_((2,2)), past interface valve 362 _((2,2)), and into second reactionchamber 320 _((2,2)), where the second material can contact the fourthmaterial. To allow such flow through reaction channel 370 _((2,2)), theinterface channel 360 ₍₂₎ can be activated so as to transform interfacevalve 362 _((2,2)) from a closed valve state to an open valve state.Under such conditions, first reaction chamber 340 _((2,2)) and secondreaction chamber 320 _((2,2)) are in fluid communication via reactionchannel 370 _((2,2)). Often, the presence, absence, or extent of anyreaction between the fourth and second materials within second reactionchamber 320 _((2,2)) can be confirmed, detected, or quantified byinspection, for example with a reader or sensor 390.

In some embodiments, the terms isolation valve and containment valve maybe used interchangeably. Similarly, the terms interface valve andreaction valve may be used interchangeably. Such valves can be actuatedor activated or otherwise controlled by any of a variety of valveoperation methods or configurations. Exemplary valve systems andtechniques which are well suited for use with embodiments of the presentinvention are described, for example, in U.S. Pat. No. 6,408,878, thecontent of which is incorporated herein by reference. Often, such valvesinclude an elastomeric portion that, when actuated, deflects into arecess. For example, FIG. 5A shows a side view or cross section of amicrofluidic device unit cell 500. The unit cell includes a firstchannel 530 and first sample chamber 540 in a first layer 581 of anelastomeric block 580, control channel 550 and via 511 a in a secondlayer 582, and a reaction channel 570 in a third layer 583. An isolationvalve 532 can be actuated, so as to inhibit or prevent flow throughfirst channel 530. Actuation of isolation valve 532 can involve thedeflection of an elastomeric portion 532 a into a recess 531 of firstchannel 530. Fourth layer 584 includes interface channel 560. FIG. 5Bshows another side view or cross section of microfluidic device unitcell 500. The unit cell includes a sample chamber 520 in a first layer581, a control channel 550 and via 531 b in a second layer 582, and asecond channel 510 in a third layer 583. Isolation valve 512 can beactuated, so as to inhibit or prevent flow through second channel 510.Actuation of isolation valve 512 can involve the deflection of anelastomeric portion 512 a into a recess 511 of second channel 510. FIG.5C shows a side view or cross section which is orthogonal to the sideviews of FIGS. 5A and 5B. As depicted here, actuation of control channel550 can operate to activate both isolation valve 532 and isolation valve512. For example, by changing the pressure of fluid within controlchannel or containment line 550, it is possible to simultaneouslydeflect a first elastomeric portion upward into first channel 530 and asecond elastomeric portion downward into second channel 510. Optionallythis can result in the containment or isolation of a first materialwithin a first chamber and a second material within a second chamber. Asshown in FIG. 5C, first channel 530 and second channel 510 each presenta rectangular cross section. In some instances, either or both of thesechannels can present a dome shaped cross section, where the dome isupright with regard to first channel 530 and inverted with regard tosecond channel 510.

FIG. 6A illustrates a microfluidic device 699 according to embodimentsof the present invention. Materials can be delivered from wells 605toward elastomeric block 608 through passages or routing lines 601. FIG.6B depicts microfluidic device 699 in a plan view. Microfluidic device699 includes a substrate 600 with integrated pressure accumulator wells601 and 602, each having a receptacle 603, 604 that contains a valve,such as a check valve. Microfluidic device 699 also includes one or morewells 605 for receiving materials such as samples or reagents, and oneor more channels or routing lines disposed between wells 605 and anelastomeric block location 607 of substrate 600. An elastomeric block608 can be coupled with substrate 600 at elastomeric block location 607.Elastomeric block can include one or more layers of elastomeric materialhaving microfabricated recesses or channels formed therein. Elastomericblock 608 can be coupled with substrate 600 in any of a variety of ways.For example, the elastomeric block can be attached or bonded with thesubstrate. In some cases, the block is directly bonded to the substrate.In some cases, the block is coupled with the substrate without the useof an adhesive. In some cases, the block is coupled with the substratewith an adhesive. Within elastomeric block 608 are one or more channelsin fluid communication with one or more vias 614, which in turn providefluid communication between the elastomeric block channels and thesubstrate channels. Hence, the substrate channels can provide fluidcommunication between wells 605 and channels within the elastomericblock.

Accumulator wells 601, 602 often include valves 611, 612, respectively,which can be check valves for introducing and holding gas of fluid underpressure into accumulator chambers 615 and 616. Valves 611 and 612 aresituated inside of receptacles 604 and 603, respectively, which can keepliquid, when present in accumulator chambers 615 and 616, fromcontacting valves 611 and 612. In some embodiments, valves 611 and 612may be mechanically opened by pressing a shave, pin or the like, withina check valve to overcome a self closing force of the check valve,thereby permitting release of pressure from the accumulator chamber, orreducing fluid pressure contained within the accumulator chamber.

Substrate 600 and associated components may be fabricated from polymers,such as polypropylene, polyethylene, polycarbonate, high-densitypolyethylene, polytetrafluoroethylene PTFE or Teflon®, glass, quartz,transparent materials, polysilicon, metals, such as aluminum, or thelike. Any of a variety of gases, liquids, or fluids can be introducedinto accumulator chambers 615 and 616. In some cases, valves 611 and 612can be actuated to release fluid pressure otherwise held inside ofaccumulator chambers 615 and 616. Optionally, a portion of substrate 600beneath the elastomeric block region 607 can be transparent. In somecases, the portion may be opaque or reflective. Accumulator chambers 601and 602 can be in fluid communication with channels contained inelastomeric block region 607, and ultimately, with channels contained inelastomeric block 608. Accumulator operation is described in U.S. PatentPublication No. 2007/0196912, the content of which is incorporatedherein by reference. In some cases, operation of a channel, such as acontrol channel 150 as shown in FIG. 1, can be modulated or controlledby an accumulator.

FIG. 7 illustrates a microfluidic device 700 according to embodiments ofthe present invention. Device 700 includes a carrier 710 coupled with achip or block 750. Carrier or frame 710 includes a plurality of routinglines 712 configured to allow flow from carrier wells toward chip 750.For example, routing lines disposed on the “S” side of the carrier canprovide for the passage of sample, and routing lines disposed on the “R”side of the carrier can provide for the passage of reagent. In somecases, chip 750 can also include a plurality of routing lines 752. Forexample, routing lines on the chip can provide material transport on thechip from location A to location B, and from location C to location D.In this way, a portion of the samples loaded onto the carrier can betransported to location E of the chip, and another portion of thesamples loaded onto the carrier can be transported to locations D and Bof the chip, such that some sample is loaded at one side of the block,and some sample is loaded at an opposing side of the block, as furtherdiscussed herein with reference to FIG. 3.

FIG. 8 illustrates a microfluidic device 800 according to embodiments ofthe present invention. Device 800 includes a carrier 810 coupled with achip or block 850. Carrier or frame 810 includes a plurality of routinglines 812 configured to allow flow from carrier wells toward chip 850.For example, routing lines disposed on the “S” side of the carrier canprovide for the passage of sample, and routing lines disposed on the “R”side of the carrier can provide for the passage of reagent. As shown inthis illustration, 24 samples loaded into wells at zone S₁ flow to theleft side of the chip (upper half), 24 samples loaded into wells at zoneS₂ flow to the upper side of the chip (left half), 24 samples loadedinto wells at zone S₃ flow to the upper side of the chip (right half),and 24 samples loaded into wells at zone S₄ flow to the right side ofthe chip (upper half). Thereafter, through another set of routing linesoptionally disposed at or within the elastomeric block, the S₁ samplesflow to the left side of the chip (lower half), the S₂ samples flow tothe left side of the chip (upper half), the S₃ samples flow to the rightside of the chip (upper half), and the S₄ samples flow to the right sideof the chip (lower half). Further, 24 reagent portions loaded into wellsat zone R₁ flow to the left side of the chip (lower half), 24 reagentportions loaded into wells at zone R₂ flow to the lower side of the chip(left half), 24 reagent portions loaded into wells at zone R₃ flow tothe lower side of the chip (right half), and 24 reagent portions loadedinto wells at zone R₄ flow to the right side of the chip (lower half).Thereafter, through another set of routing lines optionally disposed ator within the elastomeric block, the R₁ reagent portions flow to thelower side of the chip (left half), the R₂ reagent portions flow to thelower side of the chip (left half), the R₃ reagent portions flow to thelower side of the chip (right half), and the R₄ samples flow to thelower side of the chip (right half). Hence, routing lines on or in thechip can provide material transport on or through the chip from onelocation to another. In this way, a portion of the samples loaded at oneend of the carrier (e.g. the “S” end) can be transported such that somesample is loaded at one side of the block, and some sample is loaded atan opposing side of the block, as further discussed herein withreference to FIG. 3.

In some embodiments, microfluidic devices may contain blind flowchannels which include a region that functions as a reaction chamber orreaction site. Blind flow, or blind fill, can refer to the filling of adead-end tube or lumen with a liquid where a head of gas is pushed infront of the liquid bolus, and where that head of gas is vented orotherwise released from the lumen, allowing the dead-end lumen to fillfully with the liquid. In some embodiments, polydimethylsiloxane (PDMS)can be used as an elastomeric material. PDMS is sufficiently gaspermeable that liquid pressurized at a few psi can drive the gas out ofthe channels, leaving them completely filled with liquid.

Table 1 provides an exemplary experimental design where variousmaterials can be loaded or introduced into a microfluidic device thatincludes four unit cells. According to this table, sample can be flowedthrough first channels and reagent can be flowed through secondchannels. It is understood that alternatively, reagent can be flowedthrough first channels and sample can be flowed through second channels.Embodiments of the present invention encompass techniques were sample isflowed through a set of first channels and a set of second channels, andreagent is flowed through a set of first channels and a set of secondchannels.

TABLE 1 channel material chamber first channel DNA sample from firstchambers 330₍₁₎ person A 340_((1, 1)), 340_((1, 2)) first channel DNAsample from first chambers 330₍₂₎ person B 340_((2, 1)), 340_((2, 2))second channel disease X gene second chambers 310₍₁₎ primers/probes320_((1, 1)), 320_((2, 1)) second channel disease Y gene second chambers310₍₂₎ primers/probes 320_((1, 2)), 320_((2, 2))

Table 2 shows the mixtures occurring in the microfluidic device reactionchambers, and the experimental inquiries which can be answered, forexample, by conducting a PCR reaction where the sample contains patientDNA and the reagent contains an oligonucleotide primer and probe set.

TABLE 2 reaction chamber materials mixed inquiry second chamber DNAsample from person A person A has gene 320_((1, 1)) disease X geneprimer/probe for disease X? second chamber DNA sample from person Aperson A has gene 320_((1, 2)) disease Y gene primer/probe for diseaseY? second chamber DNA sample from person B person B has gene320_((2, 1)) disease X gene primer/probe for disease X? second chamberDNA sample from person B person B has gene 320_((2, 2)) disease Y geneprimer/probe for disease Y?

It is understood that any of a variety of materials may be mixed orreacted in according to embodiments of the present invention. Forexample, genotyping applications may involve detecting the presence orabsence of a target in a sample. Gene expression applications mayinvolve measuring or quantifying amounts of materials contained in asample. Such applications may benefit from the enhanced mixing functionprovided by embodiments of the present invention. Further, microfluidicdevices and methods can be used to crystallize a protein. In oneembodiment a method includes providing a microfluidic device having afirst chamber having a dimension between 1000 μm and 1 μm, a secondchamber having a dimension between 1000 μm and 1 μm, and one or moreflow or control channels each having a dimension between 1000 μm and 1μm. The first and second chambers can be in fluid communication witheach other through a channel. A valve can be disposed along a channelwhich, when actuated to open or close, controls fluid communicationbetween the first and second chambers, or into or out of the first orsecond chamber, or both. The method can include introducing acrystallization reagent into the first chamber, introducing the proteinin a solution into the second chamber, opening a valve so that thesolution containing the protein in the second chamber becomes in fluidcommunication with the crystallization reagent in the first chamber, andclosing the valve after a period of time to interrupt fluidcommunication between the first and second chambers.

In some embodiments, a valve can be under the control of an automatedvalve actuating device, which in turn may be further under control of acomputer or processor. A multilayer microfluidic device can include atleast one elastomeric layer, and a valve can include a deflectablemembrane. In some cases, a deflectable membrane of a valve can bedeflectable into one or more channels to control fluid movement alongthe channels. Multiple elastomeric membranes may be bonded or adheredtogether to form an elastomeric block. In some cases, portions ofchannels or chambers can overlap with portions of other channels orchambers at an overlap region. Such channels or chambers can be in fluidcommunication through a via located at the overlap region.

FIGS. 9-13 disclose additional aspects of microfluidic systems andmethods according to embodiments of the present invention. FIG. 9 showsa photomicrograph, with scale bar, of an exemplary microfluidic device.FIGS. 10-12 each show reader images of exemplary microfluidic devicesand experimental results provided by the devices. FIG. 13 illustrates anexemplary microfluidic device, showing channel connections between inputwells and an elastomeric block. As shown in FIG. 13, a microfluidicsystem can include a microfluidic device having a multiwell plate,optionally referred to as a carrier or frame. Often, the multiwell plateincludes a plastic material, such as an injection molded plastic. Themultiwell plate can include channel connections or passages forproviding fluid communication between input wells and an elastomericblock. U.S. Patent Application No. 61/030,887 filed Feb. 22, 2008,(Integrated Carrier for Microfluidic Device) which is incorporatedherein by reference, describes carrier or frame configurations which aresuitable for use with microfluidic systems and methods of the instantapplication. FIG. 14 shows further aspects of microfluidic systems andmethods according to embodiments of the present invention.

Additional embodiments of the invention include methods for detectingnucleic acid analytes through their interactions with a nucleic acidprobe, such as a hydrolysis probe, a hairpin probe, a padlock probe(PLP) or a hybridization probe. The methods of the invention combine thefeatures of using the high throughput microfluidic device as describedherein, labeled nucleic acid probes, and homogenous assays to detectand/or quantify nucleic acid analytes with high PCR and probespecificity. Certain methods described herein may allow for thedetection of low copy number nucleic acid analyte per cell, have lowfluorescence background yielding a high signal to noise ratio. Thehomogeneous assays of the invention may have a dynamic range of at leastabout 3 orders of magnitude, more often at least about 4, even moreoften at least about 5, even more often at least about 6, often at leastabout 7, and sometimes at least about 8 orders of magnitude.

According to an embodiment of the invention, the detection and/orquantification of a plurality of nucleic acid analytes from a sample maygenerally be carried out by obtaining a pre-amplified sample, aliquotingthe sample and distributing the pre-amplified sample into reactionchambers of a microfluidic device containing the appropriate buffers,primers, probes and enzymes, performing a homogenous assay for thetarget nucleic analytes of interest, and querying the aliquots for thepresence of nucleic acid analytes.

In a first embodiment, a sample is obtained which is suspected ofcontaining the target nucleic acid analyte of interest. The sample maybe first reversed transcribed into cDNA and subjected to a preliminaryamplification reaction to generate a pre-amplified sample. In thepreliminary amplification reaction, the reverse transcribed sample issubjected to 14 cycles of PCR in order to increase the nucleic acidanalytes by about 16,000 fold.

In a second embodiment, aliquots of the pre-amplified sample aredistributed into separated compartments of a microfluidic device andcombined with the appropriate reagents. In particular, the aliquot mayhave a volume of in the range of about 1 picoliter to about 500nanoliters, more often in the range of about 100 picoliters to about 20nanoliters, even more often in the range of about 1 nanoliter to about20 nanoliters, and most often in the range of about 5 nanoliters toabout 15 nanoliters. The reagents may include a labeled nucleic acidprobe, PCR primers (e.g., forward primers and reverse primers), athermostable DNA polymerase, GT buffer, an aqueous buffer, magnesiumchloride and deoxynucleotide truphosphates, and may also include othernon-reactive ingredients. In a specific aspect, a pre-sample mix may beprepared which may include TaqMan Universal PCR master Mix,AmpliTaq-Gold (about 5 units/μl), 20×GT buffer, and H₂O. The pre-samplemix may be combined with the nucleic acid of interest, and appropriateprimers.

In one aspect of the invention, a 1×GT buffer may contain betaine in arange of about 0.1 M to about 0.8 M, BSA in a range of about 1 mg/ml toabout 4 mg/ml, glycerol in a range of about 1% to about 5%, PEG 20,000in a range of about 1% to about 5%, PEG MME550 in a range of about 0.05%to about 5%, MME5000 in a range of 1% about to about 5%, Superblock® inPBS in a range of about 1% to about 15%, Superblock® T20 in a range ofabout 1% to about 10%, and Tween 20 in a range of 0.1% about to about3%. In a specific aspect, the 1×GT buffer may contain about 0.4 Mbetaine, 2 mg/ml BSA, about 2.5% glycerol, about 2% PEG 20,000, about 1%PEG MME550, about 2.5% MME5000, about 10% Superblock® in PBS, about 5%Superblock® T20, and about 0.5% Tween 20. In a more specific embodiment,the 1×GT buffer may contain about 0.4 M betaine, 4 mg/ml BSA, about 5%glycerol, about 2% PEG 20,000, about 1% PEG MME550, about 2.5% MME5000,about 10% Superblock® in PBS, about 10% Superblock® T20, and about 1%Tween 20.

In another aspect of the invention, a 20×GT buffer may be prepared andmay be diluted to a final concentration of 1× when used in the dynamicarrays. For example, a 20×GT buffer may include betaine in a range ofabout 1M to about 10M, BSA in a range of about 5 mg/ml to about 15mg/ml, and Superblock® T20 (in TBS) in a range of about 20% to about65%. In a particular aspect, the GT buffer may include about 5 Mbetaine, about 10 mg/ml BSA, and about 57% Superblock®T20 in TBS. As oneskilled in the art appreciates, the 20×GT buffer would be diluted to 1×in the final reaction mix.

The PCR primers must be sufficiently long to prime the synthesis ofextension products in the presence of the agent for polymerization. Theexact length and composition of the primer will depend on many factors,including temperature of the annealing reaction, source and compositionof the primer, proximity of the probe annealing site to the primerannealing site, and ratio of primer:probe concentration. For example,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains in the range of about 15 to about 30nucleotides, although it may contain more or fewer nucleotides. Theprimers should be sufficiently complementary to selectively anneal totheir respective strands and form stable duplexes. One skilled in theart appreciates how to select appropriate PCR primer pairs to amplifythe target nucleic acid analyte of interest.

In a third embodiment, a homogenous assay may be performed such asreal-time PCR, for example. In this assay, the labeled nucleic acidprobe contains a stretch of nucleic acid sequences that are capable ofrecognizing 8-mer and 9-mer motifs in the target nucleic acid analyte,as described above. FRET quenching of the labeled nucleic acid probe isirrevocably eliminated when the Taq polymerase reaches the region wherethe labeled probe is annealed to the target nucleic acid analyte,recognizes the probe-template hybrid as a substrate, and subsequentlyhydrolyzes phosphodiester bonds of the probe during primer-directed DNAamplification. The hydrolysis reaction irrevocably releases thequenching effect of the quencher dye on the reporter dye, thus resultingin increasing detector fluorescence with each successive PCR cycle. Itwill be appreciated that the invention is not limited to the use ofreal-time PCR, and that other variations of PCR, described above, may beused to detect and/or quantify the analyte of interest.

The homogenous assay of the invention should not be construed to belimited to PCR-based detection methods, but may employ any method ofdetection and/or quantification to detect and/or quantify a targetnucleic acid analyte. In one aspect, PCR may be used to amplify atarget. In another aspect, other amplification systems or detectionsystems may be used, including systems described in U.S. Pat. No.7,118,910, which is incorporated herein by reference in its entirety. Ina further aspect, a detection system other than PCR may be used such asan Invader® assay (Third Wave, Madison, Wis.). In one aspect, real timequantification methods may be used to determine the quantity of a targetnucleic acid analyte present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantification method that may be used successfully with thematrix-type microfluidic devices described herein. This method ofmonitoring the formation of amplification product involves thecontinuous measurement of PCR product accumulation using a dual-labelednucleic acid probe, such as a hydrolysis probe. It will be appreciatedthat the invention is not limited to use of these probes and anytag-specific probe may be used.

In a fourth embodiment, the aliquots in the reaction chambers may bequeried for the presence of the targeted nucleic acid analyte, which isaccomplished by the use of the labeled probes. The fluorescent signalmay be monitored and quantified with fluorescence detectors, such asfluorescence spectrophotometers and commercial systems that allow themonitoring of fluorescence in PCR reactions.

Alternatively, however, the probe may be unlabeled, but may bedetectable by specific binding with a ligand which is labeled, eitherdirectly or indirectly. Suitable labels, and method for labeling probesand ligands are well known in the art, and include, for example,radioactive labels which may be incorporated by known methods (e.g.,nick translation, random priming or kinasing), biotin, fluorescentgroups, chemiluminescent groups (e.g., dioxetanes) enzymes, antibodies,gold nanoparticles and the like. Variations of this basic scheme areknown in the art, and include those variations that facilitateseparation of the hybrids to be detected from extraneous materialsand/or that amplify the signal from the labeled moiety.

It will be appreciated that specifically at least about 10, more oftenat least about 25, still more often at least about 50, even more oftenat least about 100, in some cases at least about 500 and sometimes atleast about 1000 targets may be detected using the methodology of theinvention. Thus, the method may make use of at least about 10, moreoften at least about 25, still more often at least about 50, even moreoften at least about 100, in some cases at least about 500 and sometimesat least about 1000 target-specific probes.

All publications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same.

While the exemplary embodiments have been described in some detail, byway of example and for clarity of understanding, those of skill in theart will recognize that a variety of modification, adaptations, andchanges may be employed. Hence, the scope of the present inventionshould be limited solely by the claims.

What is claimed is:
 1. A method of mixing materials in a microfluidicdevice, comprising: flowing a first material through a first flowchannel formed in a first layer of a substrate; flowing the firstmaterial through a first isolation valve, wherein the first isolationvalve is disposed along the first flow channel, comprises a firstportion of a control channel formed in a second layer of the substrateadjacent to the first layer, and is configured to control flow throughthe first flow channel into a first chamber; flowing the first materialfrom the first flow channel into the first chamber; flowing a secondmaterial through a second flow channel formed in a third layer of thesubstrate adjacent to the second layer; flowing the second materialthrough a second isolation valve, wherein the second isolation valve isdisposed along the second flow channel, comprises a second portion ofthe control channel formed in a second layer of the substrate, and isconfigured to control flow through the second flow channel into a secondchamber; flowing the second material from the second flow channel intothe second chamber; actuating the control channel so as to inhibit flowthrough the first and second isolation valves; flowing the firstmaterial from the first chamber through an interface valve into thesecond chamber, so as to mix the first material with the secondmaterial, wherein the interface valve comprises a portion of aninterface channel formed in a fourth layer of the substrate adjacent tothe third layer, and is configured to control flow between the firstchamber and the second chamber.
 2. The method of claim 1, wherein thefirst isolation valve comprises a first deflectable membrane, the secondisolation valve comprises a second deflectable membrane, and actuatingthe control channel comprises actuating the first and second deflectablemembranes.
 3. The method of claim 2, further comprising actuating aninterface channel to provide fluid communication between the firstchamber and the second chamber.
 4. The method of claim 3, wherein theinterface valve comprises a portion of an interface channel formed in afourth layer of the substrate adjacent to the third layer, and isconfigured to control flow through a reaction channel formed in thethird layer.
 5. The method of claim 3, wherein the interface valvecomprises a deflectable membrane, and actuating the interface channelcomprises actuating the deflectable membrane.
 6. The method of claim 3,comprising holding the first material in the first chamber at firstpressure and holding the second material in the second chamber at asecond pressure, prior to flowing the first material into the secondchamber.
 7. The method of claim 6, wherein the first pressure is greaterthan the second pressure.
 8. The method of claim 6, wherein the firstpressure is about 10 psi and the second pressure is about 0 psi.